The objective of the proposed research is to determine the genetic structure and organization of three major thiol proteinases in man: Cathepsins B, H and L. This information will then be used to clarify the role of specific cathepsin genes in normal and malignant human cells in vitro. Thiol cathepsins play a significant role in the regulation of cellular functions by their responsibility for intracellular protein turnover and zymogen processing events including the activation of proinsulin, proalbumin, prorenin and procollagenase. At the same time, altered thiol proteinase activity has been associated in a significant fashion with several types of human malignancy. We propose to define the genetic organization of these enzymes as a prerequisite to understanding certain mechanisms which regulate normal and neoplastic cell growth. Using the techniques of somatic cell genetics we will determine the chromosomal assignments of the genes for human cathepsins B, H and L. Cell hybrids carrying human deletion chromosomes will then be used to localize these genes to specific chromosomal segments. In order to further specify the structure of these genes and to obtain probes for studying cathepsin gene function, we will isolate cathepsin B and H cDNA sequences. These cDNA sequences will be characterized by restriction endonuclease mapping and cDNA sequencing. We will also determine the number and kinds of cathepsin B and H-like genes in the human genome to identify whether thiol cathepsin multigene families exist. Finally, we will explore the K562 erythroleukemia cell line as a possible model system for studyng abnormal cathepsin B gene expression in malignancy. Cathepsin B specific enzyme assays and molecular assays will be used to elucidate the abnormal cathepsin B activity in the human K562 leukemia cell line.